Throughout this disclosure, various technical and patent publications are referenced to more fully describe the state of the art to which this invention pertains. These publications are incorporated by reference, in their entirety, into this application.
Optimizing the stability of a given protein is one of the “holy grails” in many biotechnological and biochemical applications. Unfortunately it is extremely hard to predict the absolute stability of proteins and at present the most successful approaches are based on experimental search. Such approaches are obviously very time-consuming since they are based on experimental iterations. The development of a reliable computer-aided protein stabilization approach can be very helpful, but unfortunately none of the previous methods was able to provide a clear correlation between the absolute folding energy and the protein sequence. No computational method for automated sequence refinement has been reported.
The ability to predict physical and chemical properties of proteins, given their sequence and folded tertiary structure, is of crucial importance for the study of enzymes. Computational approaches for predicting the thermal stability of proteins, the difference of free energy between their folded and unfolded state, have yet to emerge and validate. That is, despite the progress in the development of models for studying the folding of proteins there are still major problems in predicting protein stability by either microscopic or macroscopic models. For example, there is a lack of a clear understanding of the magnitude of electrostatic contributions to thermal stability and to the overall folding free energy. Similarly, the values of the dielectric constants to be used and the contribution of the ionizable residues to the stability of a folded protein are only a few of the questions still unanswered.
Discretized continuum studies have suggested that charged and polar groups lead to destabilization of a folded protein. Other studies, however, have indicated that protein stability is far more complicated than originally thought, and that charged residues do not necessarily destabilize the protein core. Quite the opposite, they tend to increase protein stability. Even the general idea in continuum studies that internal ionizable residues tend to destabilize the protein core is now under reevaluation. Overall there is a growing realization that electrostatic energy is related to stability in general, and that electrostatic interactions usually stabilize the native states of proteins quite significantly.
However, the exact role of electrostatic interactions in protein stability is obscured by the competition between desolvation penalties, stabilization by local protein dipoles, and charge-charge interaction.